Quantitative Glycome Analysis Research Articles

Quantitative structural analysis of glycans expressed within tumors derived from pancreatic cancer patient-derived xenograft mouse models

Pancreatic ductal adenocarcinoma (PDAC) is an intractable malignancy for which novel therapeutic targets are in high demand. To uncover glycans expressed within PDAC, we previously performed glycome profiling of PDAC cell lines using lectin microarray and found that the lectin rBC2LCN with specificity to a Fucα1-2Galβ1-3 motif exhibited strong binding to a PDAC cell line (Capan-1) and to all tumor tissues derived from 69 pancreatic cancer patients. Nevertheless, no information was available as to whether glycans containing the Fucα1-2Galβ1-3 motif are expressed within PDAC. Here we used HPLC combined with MALDI-TOFMS to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from two types of patient-derived PDAC xenograft mouse models, PC3 (well-differentiated) and PC42 (poorly-differentiated). A higher percentage of highly branched and sialylated complex-type N-glycans was detected in PC42 relative to PC3. The percentage of core 1 O-glycans was higher in PC42 relative to PC3, whereas that of core 3 O-glycans was higher in PC3. Cancer-related glycan epitopes such as Lewis A and Lewis Y were detected in core 3 O-glycans of both PC3 and PC42. H-type3 containing the Fucα1-2Galβ1-3 motif was detected in Core 2 O-glycans in both models, explaining the molecular mechanism of the binding of rBC2LCN to PDAC.

Human Influenza Virus Hemagglutinins Contain Conserved Oligomannose N-Linked Glycans Allowing Potent Neutralization by Lectins

SummaryHemagglutinins (HAs) from human influenza viruses adapt to bind α2-6-linked sialosides, overcoming a receptor-defined species barrier distinct from the α2-3 specificity of avian virus progenitors. Additionally, human-adapted HAs gain glycosylation sites over time, although their biological function is poorly defined. Using quantitative glycomic analysis, we show that HAs from human pandemic viruses exhibit significant proportions of high-mannose type N-linked glycans throughout the head domain. By contrast, poorly adapted avian-origin HAs contain predominately complex-type glycans, which have greater structural diversity. Although oligomannose levels vary, they are present in all tested recombinant HAs and whole viruses and can be specifically targeted for universal detection. The positions of high-mannose glycosites on the HA of human H1N1 and H3N2 strains are conserved. Additionally, high-mannose-binding lectins possess a broad capacity to neutralize and prevent infection with contemporary H3N2 strains. These findings reveal the biological significance of HA glycosylation and therapeutic potential of targeting these structures.

Comparative Glycomic Analysis of Sialyl Linkage Isomers by Sialic Acid Linkage-Specific Alkylamidation in Combination with Stable Isotope Labeling of α2,3-Linked Sialic Acid Residues.

Sialic acids form the terminal sugars in glycan chains on glycoproteins via α2,3, α2,6, or α2,8 linkages, and structural isomers of sialyl linkages play various functional roles in cell recognition and other physiological processes. We recently developed a novel procedure based on sialic acid linkage-specific alkylamidation via lactone ring opening (aminolysis-SALSA). Herein, we have investigated an isotope labeling of α2,3-linked sialic acid residues (iSALSA) using amine hydrochloride salts. One limitation of SALSA using amine hydrochloride salts may be solved by adding only tert-butylamine (t-BA) as an acid scavenger, and comparative and quantitative glycomic analyses can be performed using iSALSA. We also developed quantitative glycomic analysis using dual isotope-labeled glycans by derivatizing with aminooxy-functionalized tryptophanylarginine methyl ester (aoWR) and iSALSA at the reducing and nonreducing end, respectively. Furthermore, we demonstrate that the amount of α2,3-linked sialoglycans in serum are altered during liver fibrosis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography MS (LC/MS) analyses. We revealed that the ratio of A33,6,6 to A3F3,6,6 was gradually decreased along with liver fibrosis progression. Therefore, these glycan alterations are potential diagnostic markers of nonalcoholic steatohepatitis (NASH) fibrosis progression.

8-plex LC-MS/MS Analysis of Permethylated N-Glycans Achieved by Using Stable Isotopic Iodomethane.

Glycosylation is an important post-translational modification of proteins. Many diseases, such as cancer, have proved to be related to aberrant glycosylation. High throughput quantitative methods have gained attention recently in the study of glycomics. With the development of high-resolution mass spectrometry, the sensitivity of detection in glycomics has largely improved; however, most of the commonly used MS-based techniques are focused on relative quantitative analysis, which can hardly provide direct comparative glycomic quantitation results. In this study, we developed a novel multiplex glycomic analysis method on an LC-ESI-MS platform. Reduced glycans were stable isotopic labeled during the permethylation procedure, with the use of iodomethane reagents CH2DI, CHD2I, CD3I, 13CH3I, 13CH2DI, 13CHD2I, 13CD3I, and CH3I. Up to 8-plex glycomic profiling was possible in a single analysis by LC-MS, and a 100 k mass resolution was sufficient to allow a baseline resolution of the mass differences among the 8-plex labeled glycans. The major advantages of this method are that it overcomes quantitative fluctuations caused by nanoESI, it facilitates a level of comparative quantitative glycomic analysis that accurately reflects the quantitative information in samples, and it dramatically shortens analysis time. Quantitation validation was tested on glycans released from bovine fetuin and model glycoprotein mixtures (RNase B, bovine fetuin, and IgG) with good linearity (R2 = 0.9884) and a dynamic range from 0.1 to 10. The 8-plex strategy was successfully applied to a comparative glycomic study of cancer cell lines. The results demonstrate that different distributions of sialylated glycans are related to the metastatic properties of cell lines and provide important clues for a better understanding of breast cancer brain metastasis.

Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags: A Probe for Quantitative Glycomic Analysis.

Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.

Quantitative Glycomic Analysis by Mass-Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags and High-Resolution Mass Spectrometry.

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS1 level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS1 peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.

Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT.

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.

Structural and quantitative evidence of \u03b12\u20136-sialylated N-glycans as markers of the differentiation potential of human mesenchymal stem cells

Human somatic stem cells such as mesenchymal stem cells (hMSCs) have the capacity to differentiate into mesenchymal tissue lineages and to alter immune regulatory functions. As such, they hold promise for use in stem cell-based therapies. However, no method is currently available to evaluate the actual differentiation capacity of hMSCs prior to cell transplantation. Previously, we performed a comprehensive glycan profiling of adipose-derived hMSCs using high-density lectin microarray and demonstrated that α2–6-sialylation is a marker of the differentiation potential of these cells. Nevertheless, no information was available about the structural details of these of α2–6-sialylated glycans. Here we used high performance liquid chromatography (HPLC) analysis combined with mass spectrometry (MS) to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from early (with differentiation ability) and late (without differentiation ability) passages of adipose tissue-derived hMSCs. Findings in these cells were compared with those from human induced pluripotent stem cells (hiPSCs), human dermal fibroblasts (hFibs) and cartilage tissue-derived chondrocytes. A higher percentage of α2–6-sialylated N-glycans was detected in early passage cells (24–28 % of sialylated N-glycans) compared with late passage cells (13–15 %). A major α2–6-sialylated N-glycan structure detected in adipose-derived hMSCs was that of mono-sialylated biantennary N-glycan. Similar results were obtained for the cartilage tissue-derived chondrocytes, Yub621c (28 % for passage 7 and 5 % for passage 28). In contrast, no significant differences were observed between early and late passage hMSCs with respect to α2–6-sialylated O-glycan percentages. These results demonstrate that levels of α2–6-sialylated N-glycans, but not O-glycans, could be used as markers of the differential potential of hMSCs.

Quantitative glycome analysis of N-glycan patterns in bladder cancer vs normal bladder cells using an integrated strategy.

Diagnosis of bladder cancer, one of the most common types of human cancer, at an early (nonmuscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy in general is closely associated with alterations of glycan expression. Glycosylation status, particularly global glycomes, in bladder cancer has not been well studied. We integrated lectin microarray and mass spectrometry (MS) methods to quantitatively analyze and compare glycan expression in four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed using lectin microarray analysis and confirmed by lectin staining and lectin blotting. Associations of glycopatterns with diverging stages were evaluated by lectin histochemistry on tissue microarrays. N-Glycans were derivatized by amidation of sialylated glycans with acetohydrazide and reductive amination with the stable isotope tags [(12)C6]- and [(13)C6]-aniline, and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). N-Glycan biosynthesis-associated proteins were quantitatively analyzed by a stable isotope labeling by amino acids in cell culture (SILAC) proteomics method, which revealed significant differences in expression of 13 glycosyltransferases and 4 glycosidases. Our findings indicate that sialyl Lewis X (sLe(x)), terminal GalNAc and Gal, and high mannose-type N-glycans were more highly expressed in bladder cancer cells and tissues than in normal cells. Bladder cancer cells showed high expression of core-fucosylated N-glycans but low expression of terminally fucosylated N-glycans. Each of these glycome changes may be directly related to bladder cancer progression.

Analytical Platform for Glycomic Characterization of Recombinant Erythropoietin Biotherapeutics and Biosimilars by MS

Erythropoietin is a therapeutic glycoprotein that stimulates red blood cell production. The quality, safety and potency of recombinant erythropoietins are determined largely by their glycosylation. Small variations in cell culture conditions can significantly affect the glycosylation, and therefore the efficacy, of recombinant erythropoietins. Thus, detailed glycomic analyses are necessary to assess biotherapeutic quality. We have developed a platform for qualitative and quantitative glycomic analysis of recombinant erythropoietins. The platform was used to profile native N-glycans from three production batches of darbepoetin alfa (also known as NESP), a common form of recombinant erythropoietin. Darbepoetin alfa was found to contain an abundance of large, multi-antennary N-glycans with high levels of sialylation, O-acetylation and dehydration. Results were verified by independent orthogonal analysis with both MALDI-TOF and nano-LC/Q-TOF MS. This platform may be applied to QC and batch analysis of not only recombinant erythropoietin, but also other complex, glycosylated biotherapeutics and biosimilars.

Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2-3 to α-2-6, and the expression of α-1-2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a "glycome shift" upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs.

A general approach for the purification and quantitative glycomic analysis of human plasma

The development of a general method for the purification and quantitative glycomic analysis of human plasma samples to characterize global glycosylation changes shall be presented. The method involves multiple steps, including the depletion of plasma via multi-affinity chromatography to remove high abundant proteins, the enrichment of the lower abundant glycoproteins via multi-lectin affinity chromatography, the isotopic derivatization of released glycans, and quantitative analysis by MALDI-TOF MS. Isotopic derivatization of glycans is accomplished using the well-established chemistry of reductive amination to derivatize glycans with either a light analog ((12)C anthranilic acid) or a heavy analog ((13)C(7) anthranilic acid), which allows for the direct comparison of the alternately labeled glycans by MALDI-TOF MS. The method displays a tenfold linear dynamic range for both neutral and sialylated glycans with sub-picomolar sensitivity. Additionally, by using anthranilic acid, a very sensitive fluorophore, as the derivatization reagent, the glycans can be analyzed by chromatography with fluorescence detection. The utility of this methodology is highlighted by the many diseases and disorders that are known to either show or be the result of changes in glycosylation. A method that provides a generic approach for sample preparation and quantitative data will help to further advance the field of glycomics.